Tuberculosis is the most common opportunistic infection in patients with AIDS. A synergism between the etiologic agent of tuberculosis, Mycobacterium tuberculosis, and HIV is widely acknowledged. M. tuberculosis is a facultative intracellular organism that infects macrophages residing in a specialized phagosme that does not follow the default pathway of maturation into the phagolysosome. Our central objective is to find new methods to counteract or overcome the intracellular trafficking block imposed by mycobacteria. We have evidence that, in cells incubated under conditions that stimulate the autophagic pathway, mycobacterium-containing phagosomes acidify and acquire markers of lysosomal compartments resulting in a loss of bacterial viability. Thus, autophagy, which can be pharmacologically induced, is the topic of the present proposal in the context of inhibiting M. tuberculosis intracellular growth. Our main goal is to study the role of autophagy in phagosomal biogenesis and killing of intracellular M. tuberculosis in order to develop future strategies for tuberculosis treatment. Since our preliminary results indicate that induction of autophagy, by amino acid deprivation or by pharmacological means, results in an override of the maturation block imposed by mycobacteria, we will further prove these relationships by assessing the effect of LC3 and Rab24 overexpression, as these proteins are either known or are suspected to control important stages in the autophagic pathway. Specifically, we will determine the role of these proteins on maturation of the mycobacterial phagosome specialized autophagy regulators that may control the PI3K hVPS34 activity on the properties of mycobacterium-containing phagosomes and determine whether they promote antimycobacterial activity. The proposed experimental approaches will allow us to prove autophagy can counteract the intracellular trafficking block imposed by mycobacteria and find new modalities for eliminating pathogenic mycobacteria. Thus, defining the molecular mechanism by which autophagy affects intracellular mycobacteria will allow us to find new strategies to control the persistence, latency, and transmission of tuberculosis.